Since the emergence of tissue engineering as a scientific field, collagen has been used as a biomaterial for tissue engineered scaffolds. As a protein found ubiquitously throughout the human body, collagen has very low immunogenicity and antigenicity. Collagen can be molded into scaffolds of various sizes and morphologies in both the solid and liquid/gel state; some examples of this include electrospun scaffolds, lyophilized collagen scaffolds, and collagen hydrogel scaffolds. Methods used to form such collagen scaffolds allow the collagen scaffolds to be tailored to have a desired porosity and permeability. Furthermore, collagen has been shown to be an influential factor in orchestrating the adhesion, migration, and proliferation of cells during tissue growth. In addition, collagen scaffolds also evoke a less disruptive immune response than many synthetic polymers.
However, collagen does have one major weakness as a tissue engineered scaffold material—its lack of mechanical strength. Because of this deficiency, untreated collagen is rarely used as the sole material in the fabrication of tissue engineered scaffolds. There are two approaches commonly utilized to circumvent this problem. The first involves using a second biomaterial in conjunction with collagen to create a collagen blend. A number of materials have been used, including natural polymers, synthetic polymers, carbon nanotubes, and ceramics. This approach increases the mechanical strength of the collagen scaffolds but also risks increasing immunogenicity. This approach also increases the complexity of scaffold design and post-fabrication processing steps, such as sterilization and removal of residuals.
The other approach is crosslinking collagen to augment its mechanical strength. Crosslinks are covalent bonds formed between adjacent polymer chains that increase the mechanical strength of a polymer. Such bonding can be photo-activated using ultraviolet (UV) irradiation, but UV crosslinking can denature proteins and is ineffective for thick samples because of its non-uniform penetration depth. Chemical crosslinking is often more effective; in particular, glutaraldehyde is a reagent that has been shown to achieve a high degree of crosslinking at relatively low concentrations. However, residual glutaraldehyde is extremely cytotoxic. For example, it has been shown that a little as 3 parts per million (ppm) residual glutaraldehyde can kill over 99% of fibroblasts, and glutaraldehyde is also a known carcinogen. Moreover, crosslinked tissue engineered structures often must undergo a rigorous heating process at temperatures as high as 120° C. for a period of up to about 12 hours to remove residual reagents, which can be detrimental to the desired physical and biochemical properties of collagen scaffolds. For instance, the standard heating process to remove residual glutaraldehyde from a gel scaffold can exceed the glass transition temperature of the material and cause collapse of the aerogel structure. Recently, more attention has been given to collagen blends and to alternative crosslinking agents, such as 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), genipin, and riboflavin.
However, if residual glutaraldehyde were removed using a faster and less disruptive method at a lower temperature where the resulting crosslinked natural polymer scaffolds maintained their mechanical and thermal stability, which is important for preserving scaffold functionality in downstream applications, it could significantly benefit the field of tissue engineering. As such, a need exists for a system and method of removing residual glutaraldehyde from crosslinked tissue engineered scaffolds including collagen, any other natural polymer (e.g., elastin, fibrinogen, or gelatin), or a combination thereof.